Role of SOX10 Immunohistochemical Expression in Diagnosing Triple Negative Breast Cancer and Its Correlation With Clinicopathological Features

Background: Triple-negative breast cancer (TNBC) poses a diagnostic challenge for histopathologists due to the reduced frequency of breast-specific markers. SOX10 has emerged as a useful diagnostic marker for TNBC. The aim of our study was to determine the frequency of SOX-10 immunohistochemical (IHC) expression in our cohort and assess its correlation with clinicopathological and histological features. Materials and methods: We included 72 primary TNBC cases. Specimens included tru-cut biopsies and excision specimens. We stained whole slide sections of these specimens with SOX10 antibody and calculated its frequency (%) of expression and H-score. We applied the chi-square test to assess the correlation between SOX10 expression and clinicopathological and histological features such as the patient's age, specimen type, tumor size, histological type, histological grade, nuclear pleomorphism, mitotic count, tumor-infiltrating lymphocytes (TILs), necrosis, calcification, lymphovascular invasion (LVI), lymph node involvement, T stage, and N stage. Results: SOX10 expression was observed in 42 (58.3%) cases with a median H-score of 57.5. The expression was significantly higher in tru-cut biopsy specimens as compared to excision specimens (73.5 vs 41.7%) and TILs negative tumors as compared to TILs positive tumors (64.3% vs 27.3). Metaplastic carcinoma showed reduced expression when compared with non-metaplastic tumors (35.7% vs 63.8%), but statistical significance was not achieved. No correlation was observed with the patient's age, tumor size, histological type, histological grade, nuclear pleomorphism, mitotic count, necrosis, calcification, LVI, lymph node involvement, T stage, and N stage. Conclusion: SOX10 was expressed in more than half of the TNBC cases of our study which not only highlights its diagnostic utility but advocated its application in combination with other breast-specific markers. The expression didn’t correlate with the majority of clinicopathological and histological features, but correlation with tru-cut biopsy specimens and absence of TILs draws attention towards possible roles of proper fixation and host immunity, respectively.


Introduction
Breast cancer (BC) is the most common malignancy worldwide, accounting for 11.7% of all cancers [1].Developing countries of Asia with historically low incidence rates have seen a rapid rise in BC incidence during the last few decades.Pakistan has the highest age-standardized incidence rate in South Asia and the highest age-standardized mortality rate in Asia [2][3][4].Triple-negative breast cancer (TNBC) is a molecularly heterogeneous subgroup of BC that is characterized by negative expression for three biomarkers namely estrogen receptors (ER), progesterone receptors (PR), and human epidermal growth factor receptor-2 (HER-2).TNBC comprises 10-45% of all BC [5][6][7][8][9].TNBCs are further divided into basal-like BC (BLBC) and nonbasal-like BC (NBLBC) [10].In Asian countries, it presents more commonly in younger age, with larger size and high histological grade [3,4].TNBC bears a higher risk of visceral metastasis and demonstrates a poor prognosis [11].Metastatic progression is the most significant cause of mortality in BC patients [8].In addition, these patients have an increased risk of developing second primary carcinoma of other organs during the first five years of diagnosis [12].
In patients with metastatic disease, optimum treatment and management rely on correct diagnosis, therefore, all surgically accessible metastatic tumors (suspected BC) should be biopsied for confirmation of breast origin and reassessment of biomarkers [8].Determining the primary tumor in metastatic tumor biopsies is a routine task for histopathologists.Clinical history is of paramount importance and aid but in some tumors either it is not provided with the specimen, metastasis is the initial presentation, or primary BC is occult [13,14].Due to the diversity and overlapping of morphological features, the distinction is not possible on routine hematoxylin and eosin (H&E) examination.Metastatic BC can morphologically mimic carcinoma of the lung, gynecological, and gastrointestinal tracts [14].In cases of known primary BC, a rereview of slides for comparison of morphological features of both primary and metastatic tumors is performed, however, immunohistochemical (IHC) markers are routinely applied in all metastatic cases for confirmation of the primary site.Breast-specific IHC markers including GATA3, mammoglobin, and GCDFP15 are generally helpful in overall BC cases but their expression is reduced in TNBC [8,9,[15][16][17].Moreover, the IHC expressions of these markers are not completely concordant between primary and metastatic tumors of the same case [8,9,13,14,16].Some of the biomarker-positive primary tumors lose biomarker expression after metastasis with a discordance rate of up to 31% [8].The sensitivity of these markers improves when applied in combination [15,16].
In this study, we aimed to determine the frequency of SOX10 expression in TNBC.We also attempted to find a correlation between SOX10 expression and clinicopathological features.

Materials And Methods
This study was approved by the institutional ethics review committee of the Aga Khan University Hospital (2020-5179-14268).The surgical pathology database was searched for primary TNBC cases diagnosed between 2019 and 2022 at the histopathology section of the Department of Pathology and Laboratory Medicine, Aga Khan University Hospital.Cases in which formalin fixed paraffin embedded tissue blocks were not available, poorly fixed specimens, and post neo-adjuvant specimen were excluded.A total of 72 primary TNBC cases were included in the study.Clinicopathological data regarding the patient's age and gender was collected from the patient's medical records.Histological features including specimen type, histological type, histological grade, nuclear pleomorphism, mitotic count, necrosis, calcification, lymphovascular invasion (LVI), tumor-infiltrating lymphocytes (TILs), tumor size, tumor stage (T stage), lymph node status and nodal stage (N stage) were recorded from pathology reports.
IHC staining of paraffin-embedded tissue sections was carried out using the Dako EnVision System (Agilent Technologies, Santa Clara, CA), following the manufacturer's protocols.Four-micron thick sections were taken from formalin-fixed paraffin-embedded tissue blocks and placed on glass slides.High pH buffer Dako PT Link (Agilent Technologies, Santa Clara, CA) was used for deparaffinization, rehydration, and epitope retrieval in the pretreatment process.EnVision FLEX peroxidase-blocking reagent (Agilent Technologies, Santa Clara, CA) was applied first on the slides for five minutes and then the slides were washed with a wash buffer.The primary antibody used was an anti-SOX10 mouse monoclonal antibody (Clone 55k-2, diluted at 1:100, Santa Cruz Biotechnology, Santa Cruz, CA).Primary antibody was applied on slides, incubated for 20 minutes, and then washed with wash buffer.EnVision FLEX/HRP (secondary antibody) was then applied, incubated for 20 minutes, and subsequently washed.EnVision FLEX DAB (diaminobenzidine)+chromogen diluted in EnVision FLEX substrate buffer was then applied for five minutes and subsequently washed.
Counter-staining was done with hematoxylin.Finally, the slide was dehydrated (graduated alcohol to xylene).Lastly, a slide was mounted with DPX (dibutylphthalate polystyrene xylene) and a cover slip was applied.Known positive cases of malignant melanoma or peripheral nerve sheath tumors were used as positive external control and tonsil tissue as negative external control which were run simultaneously using the same protocol.
The slides were then microscopically examined by two pathologists for estimation of SOX10 expression in tumor cells.IHC expression of SOX-10 was assessed in the nucleus of tumor cells.The H-score was used for the interpretation of IHC expression of SOX10 which was obtained by multiplying the percentage of positive tumor cells by the staining intensity of tumor cell nuclei (0; no staining, 1; weak, 2; moderate, and 3; strong).SOX10 expression was considered positive if the H-score was ≥10 [19].The clinicopathological features of the patients are summarized in Table 1.The patient's age ranged from 23-80 years.The median age was 45 years, and the mean ± SD was 45.2 ± 12.6.The majority of the patients aged between 40 and 60 years.Almost half of the specimens were tru-cut biopsies.Invasive carcinoma of no special type (ICNST) was the most common histologic type of breast carcinoma followed by metaplastic carcinoma.More than 80% of the tumors were histological grade III.In excision specimens, tumor size ranged from 1.1-12.5 cm.The median size was 3.5 cm and the mean ± SD was 4.3 ± 2.4.The majority of the tumors that underwent excision and lymph node excision, qualified for T stage T2 and N stage N0, respectively (Table 1).

Clinicopathological and histological features Expression
Patient's age groups  Positive SOX10 expression was observed in 42 (58.3%)cases (Figure 1).The median cumulative H-score was 57.5.

FIGURE 1: SOX10 IHC expression.
(A) Intermediate view and (B) high-power view of tumor cells demonstrating moderate to strong nuclear expression for SOX10 IHC stain.
The clinicopathological and histological features were correlated with SOX10 expression and summarized in Table 2.The proportion of positive SOX10 expression was significantly higher in tru-cut biopsies and in tumors with the absence of tumor-infiltrating lymphocytes.Reduced expression of SOX10 stain was observed in metaplastic carcinoma when compared with other histologic types (35.7% vs 63.8%), but this difference was not statistically significant (Table 2).A greater proportion of SOX10 positive expression was observed in patients younger than 40 years, tumors having histologic grade II, moderate nuclear pleomorphism, and absence of necrosis, however, statistical significance was not observed (Table 2).

Discussion
SOX10 was first described in 2008 as a pan-schwannian and melanocytic marker when its expression was reported in all cases of neurofibroma, schwannoma, myoepithelioma of salivary gland, and 97% cases of melanoma [24].Further studies also observed SOX10 expression in other tumors including clear cell sarcoma, granular cell tumors, salivary gland tumors with myoepithelial differentiation, gastrointestinal stromal tumors, and gliomas [14,[25][26][27][28].Among carcinoma, SOX10 expression has been most frequently observed in salivary gland carcinoma and breast carcinoma, along with rare expression in head and neck squamous cell carcinoma, clear cell carcinoma, lung adenocarcinoma (LA), urothelial carcinoma, and colorectal carcinoma [14,25].SOX10 is not only expressed in myoepithelial cells but also in some luminal cells of benign breast acini which serve as its internal control [7,13,20,25].
Different studies have compared SOX10 expression among different subtypes of BC using different study designs and methodologies and the expression was higher in TNBC and ER-negative (ER-) subtypes as compared to ER-positive (ER+) and HER-2 positive (HER-2+) subtypes [5,7,9,13,14,20,21,29] (summarized in Table 3).*Primary tumors of metastatic tumors showing triple negative biomarker expression were included.Thirty-nine primary tumors were TNBC and 18 were non-TNBC.The percentage of expression of IHC markers in primary TNBC and non-TNBC was not separately mentioned.

Authors
**Archived samples (tissue microarray) consisted of primary, metastatic tumors and cell lines.The breakup of the sample tested for SOX10 and p16 was not mentioned.
***Separate frequencies of primary and metastatic tumors were not mentioned.
In a study of 57 metastatic TNBC, Tozbikian et al. observed higher SOX10 expression in metastatic tumors when primary tumors were TNBC (67%) as compared to non-TNBC primary tumors (39%) [8].Keeping in view the expression of SOX10 in normal myoepithelial cells of breast and salivary gland tumors with myoepithelial differentiation, the higher expression of SOX10 in TNBC and ER-tumors can be explained by their basal-like or myoepithelial differentiation [13].
Few studies also compared SOX10 expression in non-metaplastic and metaplastic subgroups of TNBC.A higher frequency of expression was observed in the non-metaplastic as compared to the metaplastic subgroup [5,17].Similarly, we also observed a higher frequency of SOX10 expression in non-metaplastic carcinoma (63.8%) as compared to metaplastic carcinoma (35.7%).
SOX10 is not only useful for confirming breast origin of tumors at metastatic sites but it is also performed on the breast samples as part of the diagnostic workup for poorly differentiated malignant neoplasm [13].It can also be helpful in distinguishing metaplastic carcinoma from phyllodes tumor.In Cimino-Mathews et al.'s study SOX10 was positive in 46% of metaplastic carcinoma while it was negative in all 34 phyllodes tumors [5].
Most of the studies on SOX10 have been performed using tissue microarray (TMA) and only a few have used whole sections [7,8,17,20,21].In Jamidi et al.'s study where test and validation cohorts were evaluated using TMA and whole sections, respectively, the frequency of SOX10 expression was higher in whole sections which led the authors to suggest that the expression can be affected by the amount of tissue [7].However, we didn't observe any difference in expression among frequencies reported by different studies using TMA and whole sections (Table 3).In addition, we observed a significantly higher frequency of expression in tru-cut biopsies as compared to excision specimens which also contradicts the view that expression is affected by the amount of tissue.We think that the higher frequency of expression in tru-cut biopsies might be related to special care given to the proper fixation of these samples while larger specimens might face fixation issues related to delay in transportation from different parts of our country.
The expressions of GATA3 and SOX10 are not completely concordant and independent of each other since GATA3 is considered a luminal marker while SOX10 is considered a basal/myoepithelial marker [7,8,13,17].In Yoon et al.'s study (including both primary and metastatic BC), 36.6% of cases were SOX10 positive and GATA3 negative while 26.7% cases were GATA3 positive and SOX10 negative [17].In Laurent et al.'s study, 9/18 metastatic TNBC cases were positive for SOX10, however, it was the only positive breast-specific marker in 4 cases [16].
The main differentials of metastatic tumors in axillary and cervical lymph nodes are BC and LA.The differential expression of SOX10, GATA3, and TTF-1 is usually helpful in determining the tumor origin.However, one should be aware of the rare expression of TTF-1 in BC and SOX10 or GATA3 expression in LA cases [16,30].Laurent et al. observed the sensitivity and specificity of various breast-specific markers in TNBC and TTF-1 positive and TTF-1 negative LA cohorts.SOX10 was found to be the most sensitive (62.3%) and the most specific (100%) marker [16].When the combination of SOX10, GATA3, and GCDFP15 was analyzed in TNBC and TTF-1 negative LA cohorts, the sensitivity was raised to 88.4% and specificity to 95.2%.The authors suggested sequential application of SOX10, GATA3, and GCDFP15 for differentiating between TNBC and LA [16].
Another important diagnostic challenge is faced while examining axillary tumors of unknown primary origin which are cytokeratin negative, S100 positive and SOX10 positive.These tumors are considered metastatic melanoma.This IHC profile can also be observed in metaplastic BC cases.In such cases, additional IHC markers should be avoided for differentiating metaplastic BC and melanoma, such as high molecular weight keratins, p63, HMB45, MelanA, etc. [5,20].
Very few studies have correlated SOX10 expression with clinicopathological features and observed significant correlation (Table 3) [7,13,19,21,30].Positive correlation has been reported with younger age, histological grade III, clinical stage III, pT1 stage, N stage N2, necrosis, high Ki-67 index (≥20%), and basal markers' expression [21][22][23], while negative correlation has been reported with apocrine features, negative AR expression and disease-free survival [7,21].Kriegsmann et al. assessed but couldn't find any correlation with lymphocytic stroma [23].In our study, we identified significantly reduced SOX10 expression in tumors with the presence of TILs.This finding would raise the possibility of any association between SOX10 and host immunity.

Limitations
The limitation of our study was a relatively smaller sample which affected the assessment of correlation between SOX10 expression and clinicopathological and histological features.In addition, our study was limited to primary tumor samples and we didn't assess the diagnostic utility of SOX10 IHC stain in metastatic tumor samples which are more challenging cases in real practice.

Conclusions
SOX10 is a fairly sensitive marker for triple-negative breast cancer but it should always be used in conjunction with GATA3 immunohistochemical stain.The positive correlation of SOX10 immunohistochemical expression with tru-cut biopsy samples and the negative correlation with tumorinfiltrating lymphocytes point towards possible roles of proper tissue fixation and host immunity in its expression.
Committee of Aga Khan University issued approval 2020-5179-14268.Animal subjects: All authors have confirmed that this study did not involve animal subjects or tissue.Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work.Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work.Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.

TABLE 1 : Summary of clinicopathological and histological features of triple-negative breast cancer patients (n=72).
ICNST: Invasive carcinoma of no special type; IHC: Immunohistochemical. *These clinicopathological features have been assessed in excisional biopsy specimens.** N stage has been assessed in excisional biopsy cases with lymph node excision.

TABLE 2 : Comparison of clinicopathological and histological features with SOX10 IHC expression (n=72).
*These clinicopathological features have been assessed in excisional biopsy specimens.
ICNST: Invasive carcinoma of no special type; IHC: Immunohistochemical. *Outside cases received for review are excluded.**** N stage has been assessed in excisional biopsy cases with lymph node excision.